Human SIRT1 belongs to an evolutionary conserved family of genes that regulate lifespan and stress resistance in several lower organisms. A series of studies are proposed to investigate the hypothesis that SIRT1 functions in the establishment and maintenance of cellular senescence, in response to chronic cellular stress. This function of SIRT1 may be a double-edged sword that is tumor suppressive, but can also contribute to age-associated pathologies. The focus will be on two cell types in which cellular senescence can have important physiologic consequences: (1) primary human fibroblasts, in which accumulation of senescent cells can disrupt tissue architecture and function, and (2) tissue progenitor cells, in which cellular senescence can impair tissue regenerative capacity. These studies should provide important insights into a fundamental cellular process involved in cancer and aging, and suggest strategies to inhibit potential deleterious effects of cellular senescence, without compromising tumor suppression. The long-term goal is to understand the molecular processes that contribute to aging and age-related pathologies. Three Specific Aims are proposed: 1. Characterize the role of SIRT1 in regulation of cellular senescence in primary human cells. Stable knockdown of SIRT1 expression in primary human fibroblasts and bone marrow-derived tissue progenitor cells will be performed to define the role of SIRT1 in cellular senescence and cell-cycle arrest responses to prolonged cell division, chronic oxidative stress, and metabolic stress. 2. Determine the molecular pathways by which SIRT1 contributes to the establishment of cellular senescence. The establishment of cellular senescence is controlled by two parallel pathways involving tumor suppressor and cell-cycle regulatory factors. Biochemical and functional studies will be carried out to elucidate the molecular mechanisms by which SIRT1 influences these pathways. 3. Characterize the function of human SIRT1 in regulating the stability and maintenance of the senescent state. Biochemical and functional studies will be carried out to investigate the hypothesis that SIRT1 contributes to the irreversibility of cellular senescence, and that inactivation of SIRT1 in senescent cells may allow senescence reversal.